Patent Application
20130274185
The present invention relates to a pharmaceutical for pseudo-exercise therapy.
One of the goals of diabetes treatment is to prevent or inhibit progression of various chronic complications in diabetes, for example, microvascular disorders such as diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy and macrovascular disorders such as cerebrovascular disorders, ischemic heart disease, and diabetic gangrene. Diet therapy and exercise therapy are fundamental in the diabetes treatment. In addition to these therapies, drug therapy using insulin preparations, sulfonyl urea drugs and incretin-related drugs stimulating pancreatic insulin secretion, biguanide drugs having mainly extra-pancreatic action, thiazolidine-based drugs, and carbohydrate absorption inhibitors (α-glucosidase inhibitors) is used to control blood sugar levels.
The exercise therapy, when used for the treatment of diabetes, accelerates utilization of glucose and fatty acids and thereby decreases blood sugar levels, as an acute effect of exercise and at the same time, ameliorates insulin resistance as a chronic effect. In addition, an effect on weight loss, prevention of muscular atrophy or osteoporosis due to aging or lack of exercise, or amelioration of hypertension or hyperlipidemia can be recognized. As the exercise therapy, aerobic exercise is said to be effective for amelioration of insulin resistance.
Thus, the exercise therapy is one of fundamental therapies in the treatment of diabetes. It should be the first-line therapy for ameliorating insulin resistance in the treatment of insulin independent diabetes from which particularly many patients in Japan suffer. As exercise effective for the treatment, walking continuously for at least 15 minutes twice a day and three or more days a week is said to be desirable. For many diabetic patients, however, it is not easy to continue a sufficient exercise therapy.
In particular, it is difficult to apply a sufficient walking exercise therapy to old patients having deteriorated muscle strength or patients having disorders in knee joint or hip joint. Application of the exercise therapy to diabetic patients suffering also from a disease such as arrhythmia is not always safe and it may worsen the clinical conditions of diabetic patients suffering from chronic complication (for example, patients suffering from diabetic retinopathy or diabetic nephropathy). Moreover, patients in their prime or middle-aged or older patients cannot take enough time for the exercise therapy so that they cannot often complete the scheduled exercise therapy. From such viewpoints, there is an eager demand for the development of a drug therapy for mimetically carrying out an exercise therapy.
Adiponectin (J. Biol. Chem. 270, 26746-26749 (1995); J. Biol. Chem. 271, 10697-10703 (1996); Biochem. Biophys. Res. Commun. 221, 286-289 (1996); and J. Biochem. 120, 803-812 (1996)) encoded by Adipoq is adipokine having anti-diabetic action and anti-atherogenic action. It has been reported (Arterioscler. Thromb. Vasc. Biol. 20, 1595-1599 (2000)) that plasma adiponectin levels are decreased in obesity, insulin resistance, and type 2 diabetes. Administration of adiponectin to mice causes antihyperglycemic action and ameliorates insulin resistance (Proc. Natl. Acad. Sci. USA 98, 2005-2010 (2001); Nature Med. 7, 941-946 (2001); and Nature Med. 7, 947-953 (2001)), but adiponectin-deficient mice show insulin resistance and diabetes (J. Biol. Chem. 277, 25863-25866 (2002); and Nature Med. 8, 731-737 (2002)). The insulin sensitizing effect of adiponectin is presumed to appear due to an increase in fatty acid oxidation via activation of AMP-activated protein kinase (AMPK) (Nature Med. 8, 1288-1295 (2002); Proc. Natl. Acad. Sci. USA 99, 16309-16313 (2002); and Cell Metab. 1, 15-25 (2005)) and moreover via peroxisome proliferator-activated receptor α (PPARα) (Nature 405, 421-424 (2000); and J. Biol. Chem. 278, 2461-2468 (2003)).
There is a report (Nature 423, 762-769 (2003)) on the cloning of complementary DNAs (Adipor1 and Adipor2) encoding adiponectin receptor 1 (which may hereinafter be abbreviated as “AdipoR1”) and an adiponectin receptor 2 (which may hereinafter be called “AdipoR2”). AdipoR1 is much expressed in the skeletal muscle and liver, while AdipoR2 is expressed mainly in the liver. Both receptors are predicted to contain 7-transmembrane domains (Nature 423, 762-769 (2003)) but to be structurally and functionally different from G-protein coupled receptors (FASEB J. 11, 346-354 (1997); Nature 387, 620-624 (1997); EMBO J. 15, 3566-3578 (1996)). Adiponectin receptors are presumed to constitute a new receptor family.
It is indicated by using Adipor1 and/or Adipor2 knockout mice that AdipoR1 and AdipoR2 act as a main receptor for adiponectin in vivo and play an important role in the regulation of glucose and lipid metabolism, inflammation, and oxidative stress in vivo (Nature Med. 13, 332-339 (2007)). In the liver, AdipoR1 activates an AMPK pathway, while AdipoR2 activates a PPARα pathway (Nature Med. 13, 332-339 (2007)). On the other hand, insulin resistance has been reported to be associated with mitochondrial dysfunction (N. Engl. J. Med. 350, 664-671 (2004)) but the exact cause of mitochondrial dysfunction has not yet been found. The decreased adiponectin/AdipoR1 signaling may be associated with mitochondrial dysfunction, but details of adiponectin/AdipoR1 signaling have not yet been elucidated. In addition, the cascade of adiponectin/AdipoR1 signaling which has induced intracellular physiological action has been poorly elucidated.
Non-patent Document 12: Proc. Natl. Acad. Sci. USA 99, 16309-16313 (2002)
An object of the present invention is to provide a pharmaceutical that can be applied to diabetic patients as a pseudo-exercise therapeutic agent.
The present inventors have carried out an extensive investigation with a view to solving the above-mentioned problem. As a result, it has been found that adiponectin bound to adiponectin receptor 1 causes incorporation of calcium ions (Ca2+) in skeletal muscle cells. It has also been found that influx, into skeletal muscle cells, of calcium ions generated when adiponectin binds to AdipoR1 is indispensable for subsequent activation of calcium ion/calmodulin-dependent protein kinase β (CaMKKβ), activation of AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and increased mitochondria in the muscle cells. It has also been confirmed, on the other hand, that muscle-specific disruption of adiponectin receptor 1 results in decreased elevation of a calcium ion concentration by adiponectin in cells which leads to decreased activation of CaMKK, AMPK, and SIRT1 and suppression of AdipoR1 results in decreased expression and deacetylation of PGC-1α, a decreased mitochondrial content and enzymes, decreased oxidative type I myofibers and decreased oxidative stress-detoxifying enzymes in skeletal muscle.
The present inventors have paid attention to the fact that various physiological activities induced by binding of adiponectin to adiponectin receptor 1 are substantially similar to changes in skeletal muscle cells after exercise. As a result, it has been found that a pseudo-exercise therapy can be conducted by a drug therapy using a medicament containing, as an active ingredient, an adiponectin receptor 1 agonist compound and a post-exercise good muscle state achieved by applying a managed exercise therapy to patients can also be achieved by the drug therapy, leading to the completion of the present invention.
The present invention provides a pharmaceutical for pseudo-exercise therapy containing an adiponectin receptor 1 agonist compound as an active ingredient.
The present invention also provides a pharmaceutical containing an adiponectin receptor 1 agonist compound as an active ingredient and capable of changing the physiological condition of a muscle to that after exercise without putting an exercise stress.
In a preferred mode of the present invention, there is provided the above-described pharmaceutical to be used for a drug therapy as a substitute for an exercise therapy in the prevention and/or treatment of diabetics.
From another standpoint, the present invention provides a pseudo-exercise therapeutic agent containing an adiponectin receptor 1 agonist compound as an active ingredient; a pseudo-exercise effector containing an adiponectin receptor 1 agonist compound as an active ingredient; a type I muscle fiber enhancer containing an adiponectin receptor 1 agonist compound as an active ingredient; and an insulin-sensitivity enhancer based on a pseudo-exercise effect, which enhancer contains an adiponectin receptor 1 agonist compound as an active ingredient.
The present invention further provides the use of an adiponectin receptor 1 agonist compound for the preparation of the above-described pharmaceutical.
The present invention still further provides a method of conducting a pseudo-exercise therapy in mammals including humans, including a step of administering an effective amount of an adiponectin receptor 1 agonist compound to mammals including humans; a method of changing the physiological conditions of the muscle of mammals including humans to those after exercise, including the step of administering an effective amount of an adiponectin receptor 1 agonist compound to mammals including humans; and a method of increasing type I muscle fibers in the muscle of mammals including humans, including a step of administering an effective amount of an adiponectin receptor 1 agonist compound to mammals including humans.
The pharmaceutical for pseudo-exercise therapy provided by the present invention causes, when bound to adiponectin, influx of extracellular calcium ions in skeletal muscle cells and thereby causes improvement of calcium ion signaling, improvement of glucose and lipid metabolism, improvement of exercise endurance, enhancement of expression and activation of PGC-1α through AMPK/SIRT1, and improvement of the function of mitochondria and oxidative stress. These physiological changes occurring after calcium ion influx in skeletal muscle cells are substantially similar to physiological changes in skeletal muscle cells after exercise. In particular, the pharmaceutical of the present invention increases a proportion and amount of type I muscle fibers and at the same time, has an effect of enhancing insulin sensitivity so that administration of the pharmaceutical of the present invention can achieve, in muscle, effects similar to those achieved by applying good-quality exercise without actual exercise.
The pharmaceutical for pseudo-exercise therapy provided by the present invention can achieve pseudo-exercise effects through a drug therapy instead of an exercise therapy by administering it to diabetic patients who are old or have difficulty in walking and are not suited for the exercise therapy or arrhythmic patients or patients suffering from diabetic chronic complication for which an exercise therapy may be contradicted. Moreover, it can be used, as an auxiliary pharmaceutical to be used in combination with exercise therapy, for patients who cannot take sufficient time for exercise therapy. The pharmaceutical of the present invention can achieve pseudo-exercise effects in muscle without temporary or chronic muscle disorders such as damage, rupture, or inflammation of muscle fibers which will otherwise be caused by exercise and thus has an excellent characteristic.
The pharmaceutical of the present invention is characterized by that it is a pharmaceutical for pseudo-exercise therapy and contains an adiponectin receptor 1 agonist compound as an active ingredient.
The term “adiponectin receptor 1 agonist compound” as used herein means a compound which, when the compound is bound to adiponectin receptor 1, can cause all or some of various physiological actions caused by binding of adiponectin, which is an internal ligand of adiponectin receptor 1, to adiponectin receptor 1 and can exhibit physiological actions similar to those of adiponectin. This term is used as a notion embracing adiponectin itself.
As described specifically and in detail in Examples, various physiological actions which occur when adiponectin is bound to adiponectin receptor 1 are induced by incorporation of calcium ions in skeletal muscle cells which occurs first by binding of adiponectin to adiponectin receptor 1. Based on this finding, compounds having an agonist action for adiponectin receptor 1 can be screened with incorporation of calcium ions in skeletal muscle cells as an indicator.
For example, candidate compounds for adiponectin receptor 1 agonist can be screened by a method including a step of bringing test compounds into contact with cells to determine whether or not they cause intracellular influx of extracellular calcium ions and a step of selecting, from the test compounds, compounds causing intracellular influx of extracellular calcium ions as a candidate compound for adiponectin receptor 1 agonist. The term “candidate agonist compound” means a compound having at least a possibility as an agonist compound.
In a preferred mode, a candidate agonist compound for adiponectin receptor 1 can be screened by a method including a step of bringing test compounds into contact with AdipoR1-deficient cells and AdipoR1-expressing cells to judge whether or not they cause intracellular influx of extracellular calcium ions and selecting, from the test compounds, compounds causing intracellular influx of extracellular calcium ions in the AdipoR1-expressing cells without substantially causing intracellular influx of extracellular calcium ions in the AdipoR1-deficient cells as the candidate agonist compound for adiponectin receptor 1.
Candidate agonist compounds can be used as agonist compounds, but candidate agonist compounds for adiponectin receptor 1 can be screened efficiently with accuracy by a method including the following steps:
(a) bringing test compounds into contact with cells to judge whether or not they cause intracellular influx of extracellular calcium ions and selecting, from the test compounds, compounds causing the intracellular influx of extracellular calcium ions as candidate agonist compounds for adiponectin receptor 1; and
(b) bringing the candidate agonist compounds selected in the step (a) into contact with each of AdipoR1-deficient cells and AdipoR1-expressing cells to judge whether or not they cause intracellular influx of extracellular calcium ions and selecting, from the candidate agonist compounds, compounds not substantially causing intracellular influx of extracellular calcium ions in the AdipoR1-deficient cells but causing intracellular influx of extracellular calcium ions in the AdipoR1-expressing cells as an adiponectin receptor 1 agonist compound.
In these screening methods, no particular limitation is imposed on the test compound and any compounds such as oligopeptides, polypeptides, nucleic acids, saccharides, and lipids as well as low-molecular organic compounds can be provided for screening. Since a number of compound libraries having many compounds stored therein have been constructed, it is efficient to provide for screening many compounds registered in such libraries.
The kind of the cells with which the test compounds are brought into contact is not particularly limited and any somatic cell in which AdipoR1 has been expressed can be used. Preferably, cells in which AdipoR1 have been expressed abundantly, for example, skeletal muscle cells can be used. In addition, cells in which AdipoR1 has been expressed by introducing a gene encoding AdipoR1 can be used. In such a case, using, for example, oocytes are also preferred. Cells are however not limited to skeletal muscle cells or oocytes.
When the test compounds are brought into contact with the cells, it is possible to employ a common method such as a method of putting the cells in a medium adjusted to a predetermined calcium ion concentration and adding a solution of each of the test compounds having a predetermined concentration to the resulting medium, but the method is not limited to it.
As a method of detecting influx of extracellular calcium ions into cells, any method that those skilled in the art can employ can be used. In general, influx of extracellular calcium ions into cells can be detected by measuring an increased intracellular calcium ion concentration as a result of influx of calcium ions. Examples of a detecting means include a method of detecting it from fluorescence intensity by using a calcium-ion fluorescent reagent (calcium ion fluorescent probe) which scavenges calcium ions and emits fluorescence and a method of electrophysiologically detecting a current caused by influx of calcium ions into cells, and combined use of these methods. The detection method is however not limited to them.
As the calcium-ion fluorescent reagent, for example, Fluo 4-AM, Fluo 3, Fluo 3-AM, Fura 2, Fura 2-AM, Indo 1, Indo 1-AM, Quin 2, Rhod 2, and Rhod 2-AM are commercially available so that a proper one may be selected as needed, depending on the kind of the cell, kind of the test compound, or the like. For example, Fura 2-AM or the like reagent is preferably used for the measurement of the intracellular calcium ion concentration, because after incorporation into cells from the outside of the cells in the presence of a lipophilic acetoxymethyl group, it is hydrolyzed into Fura 2, coupled to calcium ions, and emits fluorescence of high intensity. It is possible to refer to, for example, Pharmacia, 26, pp. 544-546 (1990) for a fluorescent reagent for measuring an intracellular calcium ion concentration.
When the calcium ion fluorescent reagent is used, calcium ions entering cells are coupled to a calcium ion fluorescent reagent and thereby fluorescence intensity depending on the calcium ion concentration can be obtained so that the intracellular calcium concentration can be measured with fluorescence intensity as an indicator. For the measurement of fluorescence intensity, any conventional method such as observation under a fluorescent microscope or imaging can be used. It is the common practice to detect an increase in an intracellular calcium concentration from an increase in fluorescence intensity by using a calibration curve.
As a method of electrophysiologically measuring an intracellular calcium concentration, for example, as shown specifically in Examples herein, a current (for example, a current due to chlorine ions activated by the influx of calcium ions) caused by the influx of calcium ions while using cells in which, for example, adiponectin receptor 1 has been expressed can be detected by a common electrophysiological measuring means. The method of electrophysiologically measuring the concentration while using an electrical signal as an indicator is suited for high through-put screening because it can simultaneously detect signals from a large amount of cells and only an inexpensive apparatus is necessary for high-speed detection of electrical signals.
For the measurement of an intracellular calcium ion concentration, it is also preferred to use, as the cells, cells in which adiponectin receptor 1 has been expressed. For this purpose, as described specifically in Examples herein, cells in which adiponectin receptor 1 has been expressed by introducing a gene encoding adiponectin receptor 1 into oocytes or the like cells can be used. However, the kind of cells or method of introducing a gene is not particularly limited.
When from the test compounds, a test compound causing intracellular influx of extracellular calcium ions is found, this test compound can be selected as a candidate agonist compound for adiponectin receptor 1. This candidate compound is a compound having possibility of inducing all or some of various physiological actions which will be caused by adiponectin, an internal ligand of adiponectin receptor 1, when it is bound to adiponectin receptor 1.
As the AdipoR1-deficient cells, AdipoR1-deficient skeletal muscle cells can be used preferably. It is also possible to use, for example, muscle tissues or myocytes isolated and sampled from an animal (muscle-R1KO animal) produced by muscle-specifically knocking out AdipoR1 by the method described specifically in Examples herein. The method of preparing AdipoR1-deficient cells is however not limited to the above-mentioned particular method. It is needless to say that a desired cell can be prepared by any method usable by those skilled in the art.
The test compounds causing intracellular influx of extracellular calcium ions in AdipoR1-expressing cells without substantially causing intracellular influx of extracellular calcium ions in AdipoR1-deficient cells can be selected as a candidate compound for adiponectin receptor 1 agonist. This method is highly precise so that the candidate agonist compound selected using this method is a compound capable of acting as an agonist compound with high possibility.
It is possible to achieve more efficient and highly precise screening by employing, as primary screening, a step of bringing test compounds into contact with cells to judge whether they cause intracellular influx of extracellular calcium ions or not and employing, as secondary screening, a step of bringing the candidate compounds selected by the primary screening into contact with AdipoR1-deficient cells and AdipoR1-expressing cells to judge whether they cause intracellular influx of extracellular calcium ions or not. The above-mentioned two-stage screening is very useful for efficiently selecting agonist compounds for adiponectin receptor 1 from a library including many test compounds.
The pharmaceutical of the present invention containing, as an active ingredient, the adiponectin receptor 1 agonist compound obtained as described above can be administered to mammals including humans as a pharmaceutical for pseudo-exercise therapy or a pharmaceutical for changing the physiological condition of muscle to that after exercise. As the active ingredient of the pharmaceutical of the present invention, adiponectin itself is preferred but also the above-mentioned agonist compounds other than adiponectin are also preferred.
Although the present inventors do not stick to any particular theory, the pharmaceutical for pseudo-exercise therapy provided by the present invention is bound to adiponectin receptor 1 to cause influx of extracellular calcium ions in skeletal muscle cells and thereby induce improvement of calcium ion signaling, improvement of glucose and lipid metabolism, improvement of exercise endurance, enhancement of expression and activation of PGC-1α through AMPK/SIRT1, and improvement of the function of mitochondria and oxidative stress. As a result, it increases a proportion and amount of type I muscle fibers as well as effect of exercise therapy and at the same time, is effective for enhancing insulin sensitivity. Administration of the pharmaceutical of the present invention can therefore achieve, in muscle to which no exercise stress has been applied, an effect similar to that attained by application of good-quality exercise.
The term “pseudo-exercise therapy” as used herein means a therapy capable of achieving, in muscle, an effect similar to that produced by an exercise therapy without using the exercise therapy. It can achieve various exercise effects in muscle similar to those achieved by application of actual exercise, for example, amelioration of insulin sensitivity and increase in the proportion and amount of type I muscle fibers without causing extension, shrinkage, or slide of muscle fibers as caused by actual exercise.
The subject to which the pharmaceutical of the present invention can be applied is not particularly limited and the pharmaceutical can be applied to any patients or animals who require an exercise therapy. Preferably, the pharmaceutical is effective for the treatment or prevention of obesity or diabetes, preferably insulin-independent diabetes. The treatment or prevention using the pharmaceutical is expected to change the quality of muscle and improve basal metabolism, and thereby achieve amelioration of such diseases or retard the progress thereof. For diabetes which is a preferred subject, it can be applied as a substitute for the exercise therapy to be applied for the prevention and/or treatment of diabetes or it can be applied as an adjunct to the exercise therapy. It can also be applied as a substitute for rehabilitation for functional recovery after injuries or as an adjunct to such rehabilitation or introduction period thereof, or it can be applied for prevention of or recovery from muscular weakness which may occur when limbs have been partially fixed after a bone fracture or a sprain. In particular, effectiveness of an exercise therapy for diabetic patients in amelioration of insulin resistance has been recognized. It is known that an onset ratio of insulin independent type diabetes is high in Japan so that using the pharmaceutical of the present invention for insulin independent diabetic patients or patients suffering from metabolic syndrome as an onset risk group of diabetes is a preferred mode.
No particular limitation is imposed on the administration route of the pharmaceutical of the present invention and it can be administered orally or parenterally, depending on the kind of the active ingredient. Examples of pharmaceutical compositions suited for oral administration include granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, and liquids and solutions. Examples of pharmaceutical compositions suited for parenteral administration include injections for intravenous administration, intramuscular administration, or subcutaneous administration, suppositories, transdermal systems, transmucosal systems, inhalations, and patches such as cataplasms and tapes. Preparations obtained as a pharmaceutical composition in dry powder form such as lyophilized product may be dissolved before use and the resulting solution may be used as an injection or infusion. For example, when an application site of the pharmaceutical of the present invention is limited to a certain position, the pharmaceutical prepared as a cataplasm or tape exhibiting transdermal absorption can be applied to a position, for example, a muscle around a fractured or sprained site to promote recovery of the muscle.
For the preparation of the pharmaceutical composition, additives in solid or liquid form can be used. An additive may be either an organic substance or an inorganic substance. An oral solid preparation can be prepared, for example, by adding an excipient to a substance serving as an active ingredient and selected from the group consisting of compounds represented by the formula (I) or pharmacologically acceptable salts thereof, and hydrates or solvates thereof, adding a binder, a disintegrant, a lubricant, a colorant, a taste or smell corrigent, or the like to the resulting mixture as needed, and then processing the resulting mixture into tablets, coated tablets, granules, powders, or capsules in the conventional manner.
Examples of the excipient include lactose, sucrose, white soft sugar, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum Arabic, tragacanth, gelatin, Shellac, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, calcium citrate, dextrin, and pectin. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. As the colorant, any colorants acceptable as a pharmaceutical additive may be used. As the taste or smell corrigent, cocoa powder, menthol, aromatic acids, menthe oil, borneol, cinnamon powder, and the like can be used. Tablets or granules can be coated with sugar, gelatin, or the like as needed. In addition, an antiseptic, antioxidant, or the like can be added as needed.
Liquid preparations for oral administration such as emulsions, syrups, suspensions, solutions, or liquids can be prepared using commonly-used inert diluents, for example, water or plant oil. Liquid preparations may contain an adjuvant, for example, a humectant, a suspending assistant, a sweetener, a flavoring agent, a colorant, a preservative, or the like. The liquid preparation thus obtained may be poured in capsules such as gelatin capsules
Examples of the solvent or suspending agent to be used for the preparation of a pharmaceutical composition for parenteral administration, for example, injections or suppositories include water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, and lecithin. Examples of a substrate to be used for the preparation of suppositories include cacao butter, emulsified cacao butter, laurin butter, and Witepsol. No particular limitation is imposed on the preparation method of the pharmaceutical composition for parenteral administration and any method ordinarily used in this industry can be employed.
Examples of the carrier usable in preparing a pharmaceutical composition in the form of injection include diluents such as water, ethyl alcohol, Macrogol, and propylene glycol, pH regulators or buffers such as sodium citrate, sodium acetate, and sodium phosphate; and stabilizers such as sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, and thiolactic acid. The pharmaceutical composition may contain a salt, glucose, mannitol, or glycerin in an amount sufficient for preparing an isotonic solution. It may also contain a solubilizing agent, a soothing agent, or local anesthesia.
When a pharmaceutical composition in the form of ointment, for example, paste, cream or gel is prepared, ordinarily used substrates, stabilizers, humectants or preservatives may be used as needed. Ingredients may be mixed in the conventional manner to prepare a corresponding pharmaceutical composition. Examples of the substrate usable here include white petrolatum, polyethylene, paraffin, glycerin, cellulose derivative, polyethylene glycol, silicone, and bentonite. Examples of the preservative usable here include methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate. When a pharmaceutical composition in the form of a plaster is prepared, the above-mentioned ointment, cream, gel or paste can be applied to the surface of a common support by the conventional manner. Examples of the preferable substrate include a woven or nonwoven fabric made of cotton, rayon, or chemical fibers and a film or foam sheet made of soft polyvinyl chloride, polyethylene, or polyurethane.
No particular limitation is imposed on the administration amount of the pharmaceutical of the present invention and it can be selected as needed while considering the kind of the active ingredient or degree of action as an agonist and referring to the effective amount of adiponectin. When the pharmaceutical is administered orally, the dosage of it in terms of weight of the agonist compound serving as an active ingredient can be selected from a range of from about 0.01 mg to 5,000 mg a day per adult. The above-described dosage is preferably decreased or increased as needed, depending on the age, weight, sex, administration purpose, symptoms, or the like of the patient. The above-described daily dosage may be administered once or in two to four divided doses, or once in several days to several weeks after a proper interval. When the pharmaceutical is administered as an injection or infusion, the dosage in terms of the weight of the agonist compound serving as an active ingredient is from about 0.001 mg to 500 mg a day per adult.
The present invention will hereinafter be described more specifically by Examples. It should however be borne in mind that the scope of the present invention is not limited by the following Examples.
Mice used were from 8 to 10 week old at the time of the experiment. For the measurement of the exercise capacity in muscle-R1KO mice, a treadmill exercise test regimen of 15 m/min for 20 min was used and exercise endurance was assessed by dividing 20 min by the number of times a mouse failed to avoid electrical shocks. Induction of myogenic differentiation using C2C12 cells was carried out according to the method described in the following document (Nature Med. 8, 1288-1295 (2002)). By day 5, the cells had differentiated into multinucleated contracting myocytes. C2C12 myocytes were used after myogenic differentiation in all experiments.
The plasmids encoding PGC-1α and PGC-1α-2A mutants were obtained from Dr. B. M. Spiegelman and they are all as described in the following document (Proc. Natl. Acad. Sci. USA 104, 12017-12022 (2007)). The plasmids encoding PGC-1α-R13 mutants were obtained from Dr. P. Puigserver and they are all as described in the following document (Nature, 434, 113-118 (2005)). The results of statistical analysis were indicated by mean±s.e.m. A difference between two groups was assessed by using unpaired two-tailed t-test. Data including two or more groups were assessed by analysis of variance (ANOVA).
The method will next be described in detail.
(1) Generation of Muscle-R1KO Mice
Constructed was a targeting vector for Adipor1 in which a third loxP site had been introduced into the intron 2 of Adipor1 gene and the floxed (loxP-introduced) neomycin-resistant gene (neoR) had been introduced into the intron 5 of the AdipoR1 gene (
Animals transgenically expressing cre recombinase under the control of the muscle creatine kinase promoter (MCK-Cre) were obtained from Dr. C. R. Kahn of the Joslin Diabetes Center (Boston (MA), USA). These animals were backcrossed with C57Bl/6 mice more than five times.
The Adipor1lox/+ mice were intercrossed with MCK-Cre mice to generate MCK-Cre Adipor1lox/+ mice. MCK-Cre Adipor1lox/+ mice were crossed with Adipor1lox/lox mice to obtain Adipor1lox/+, MCK-Cre Adipor1lox/+, Adipor1lox/lox, and MCK-Cre Adipor1lox/lox (muscle-R1KO) mice, respectively. The MCK-Cre and Adipor1lox/lox mice were phenotypically indistinguishable; Adipor1lox/lox mice were used as controls. All experiments in this study were conducted on male littermates.
Mice were bred in a cage on a 12-hr dark-light cycle. In all the experiments, a standard feed (CE-2, CLEA Japan) having the following composition: 25.6% (wt/wt) protein, 3.8% fiber, 6.9% ash, 50.5% carbohydrate, 4% fat, and 9.2% water was used (Nature Med., 13, 332-339 (2007)).
(2) Expression Levels of AdipoR1 and AdipoR2 in Muscle-R1KO Mice
Muscle-R1KO mice were generated by crossing animals harboring a floxed AdipoR1 allele with mice that transgenically express cre recombinase under the control of the muscle creatine kinase promoter (MCK-Cre) (Mol. Cell, 2, 559-569 (1998)) (
(3) Genotyping
Genotyping of the mice was performed by using PCR. Two primers were used for AdipoR1 genotyping: the forward primer was 5′-ACA GGC ACA TAA CAT CTA AGG CA-3′ and the reverse primer was 5′-AGC ATG ATA CTG AGT TGG CCA CA-3′. The two primers used for detecting the floxed AdipoR1 gene were 5′-CGG TGT TGA CGA GGC GTC CGA AG-3′ and 5′-GGT CTT CGG GAT GTT CTT CCT G-3′. Primers used for detection of Cre were 5′-CGC CGC ATA ACC AGT GAA AC-3′,5′-ATG TCC AAT TTA CTG ACC G-3′,5′-ACA GCG TGA ATT TTG GAG TCA GAA-3′, and 5′-GCT TTG CTC AGC CTC GCT AT-3′.
(4) AMPK Phosphorylation In Vivo
To study AMPK phosphorylation in vivo, 30 μg of recombinant murine adiponectin per 10 g body weight was intravenously injected into mice through an inferior vena cava catheter (Nature Med. 8, 1288-1295 (2002)). This resulted in an increase of plasma adiponectin levels to approximately 30 μg/ml; which showed that the adiponectin dose used in this study was comparable to endogenous adiponectin levels. Mouse full-length adiponectin was generated as described in the following documents (Nature Med. 8, 1288-1295 (2002); Nature Med. 7, 941-946 (2001); Nature 423, 762-769 (2003)).
(5) Southern Blot, Northern Blot, Real-Time PCR, and Immunoblotting
Northern blot was carried out according to the method described in the following documents (Nature Med. 13, 332-339 (2007); Nature 423, 762-769 (2003)). Total RNA was prepared from cells or tissues by using Trizol (Invitrogen) according to the manufacturer's instruction. The cDNA probe template of AdipoR1 was prepared by RT-PCR using specific primers. The forward primer used was 5′-GCTGAGGAAGATCAAGCATGCCCGGTG-3′ and the reverse primer used was 5′-TTGGAAAAAGTCCGAGAGACCTTCTCTG-3′.
Southern blot was carried out according to the method described in the following documents (Nature Med. 13, 332-339 (2007); J. Biol. Chem. 281, 26602-26614 (2006)). XbaI-digested mice genomic DNA was hybridized with the probe. The cDNA probe template of AdipoR1 was prepared by RT-PCR using specific primers. The forward primer was 5′-TGTCCACCATCACAGGAAGA′-3 and the reverse primer was 5′-GCTTGCCCTTCTCCTCCA-3′.
Real-time PCR was carried out according to the method described in the following documents (Nature Med. 13, 332-339 2007); Nature 423, 762-769 (2003); J. Biol. Chem. 281, 8748-8755 (2006)). Total RNA was prepared from cells or tissues by using Trizol (Invitrogen) according to the manufacturer's instructions. Quantification of mRNA was conducted using a real-time PCR method (Nature 423, 762-769 (2003)) with slight modifications. The procedures used for immunoblotting are described in the following documents (Nature Med. 7, 941-946 (2001); J. Biol. Chem. 281, 26602-26614 (2006); J. Biol. Chem. 281, 8748-8755 (2006)). The livers or muscles were freeze-clamped in liquid nitrogen in situ (Nature Med. 7, 941-946 (2001); J. Biol. Chem. 281, 26602-26614 (2006); J. Biol. Chem. 281, 8748-8755 (2006)).
Phosphorylation and/or the protein levels of AMPKα, PGC-1α, troponin I, IRS-1, Akt, S6K1, JNK, CaMKI, MEF2, MCAD, SOD2, Catalase, Glut4, HDAC5, and FOXO1 were determined as described in the following documents (EMBO J. 19, 989-996 (2000); Nature Med. 7, 941-946 (2001); J. Biol. Chem. 281, 26602-26614 (2006); J. Biol. Chem. 281, 8748-8755 (2006); Nature 458, 1056-1060 (2009); Cell Metab. 4, 75-87 (2006); Nature 444, 860-867 (2006); Steroids 72, 422-428 (2007)). Representative data from one of 3-5-independent experiments are shown.
(6) C2C12 Cells
The mouse C2C12 myoblasts were grown in 90% Dulbecco's modified high glucose Eagle's medium containing 10% (v/v) fetal bovine serum. When the cells reached 80% confluent, the myoblasts were induced to differentiate into myocytes by replacing the medium with a low serum differentiation medium (98% Dulbecco's modified high glucose Eagle's medium, 2.5% (v/v) horse serum). The medium was changed daily. The C2C12 cells were transfected by using Pofectamine 2000 (Invitrogen) and following the manufacturer's instructions.
(7) Mitochondrial Content Assay
Mitochondrial content assays were carried out as described in the following document (Cell Metab. 4, 75-87 (2006)), with slight modifications. Quantification of the mitochondrial content was conducted by using mtDNA and MitoTracker Green probe (Molecular Probes) according to the method as described in the following document (Obes. Res. 13, 574-581 (2005)). MitoTracker Green probe preferentially accumulates in mitochondria regardless of the mitochondrial membrane potential and provides an accurate assessment of mitochondrial mass. Cells were washed with PBS and after addition of 100 nM MitoTracker Green FM (Molecular Probes), incubated at 37° C. for 30 min. Cells were harvested by using trypsin/EDTA and resuspended in PBS. Fluorescence intensity was detected with excitation and emission wavelengths of 490 and 516 nm, respectively, and the values thus measured were corrected for total protein (mg/ml).
(8) Histological Analyses
The relationship between a change in type I muscle fiber gene expression and actual muscle fiber morphology in the skeletal muscle (soleus) of muscle-R1KO mice was examined by using light microscopy and histochemical analysis. Distinction between type I and type II fibers can be made by myosin ATPase staining at different pH values. In particular, at pH 4.3, type I muscle fibers are well stained while type II fibers are not (Physiol. Rev. 80, 1215-1265 (2000)). Samples of the muscle were frozen in liquid nitrogen-cooled isopentane, and transverse serial sections were stained with modified Gomori trichrome (J. Neurol. Sci. 26, 479-488 (1975)). Cytochrome c oxidase (COX) (J. Cell Biol. 38, 1-14 (1968)) and succinate dehydrogenase (SDH) activities (A Modern Approach, Muscle Biopsy (Saunders, London, 1973)) were enzyme-histochemically analyzed and assessed.
(9) H2O2 Measurement
Plasma H2O2 levels were measured by using an Amplex Red hydrogen peroxide assay kit (Invitrogen) (J. Clin. Invest. 118, 789-800 (2008)). Mitochondrial H2O2 levels were measured by using the Amplex red-horseradish peroxidase method (Molecular probes) (Anal. Biochem. 253, 162-168 (1997)).
(10) Lipid Metabolism, Lipid Peroxidation, and Other Materials
Skeletal muscle homogenates were extracted and a tissue triglyceride content was determined by the method described in the following documents (Nature Med. 8, 1288-1295 (2002); Nature Med. 7, 941-946 (2001); J. Biol. Chem. 278, 2461-2468 (2003)) with some modifications. To investigate whether oxidative stress increased, lipid peroxidation was measured using a marker of oxidative injury as represented by plasma thiobarbituric acid reactive substance (TBARS) according to the method described in the following document (J. Biol. Chem. 278, 2461-2468 (2003)). Tissue samples were homogenized in a buffer solution containing 50 mM Tris-HCl (pH 7.4) and 1.15% KCl, and then centrifuged. The supernatant was used for the assay. The levels of lipid peroxidation in tissue homogenate and plasma were measured in terms of the amount of TBARS by using the LPO test (Wako Pure Chemical Industries). All other materials including chemical substances were obtained from the sources given in the following References (Nature Med. 8, 1288-1295 (2002); Nature Med. 7, 941-946 (2001); Nature 423, 762-769 (2003); J. Biol. Chem. 281, 26602-26614 (2006); J. Biol. Chem. 281, 8748-8755 (2006); J. Biol. Chem. 279, 30817-30822 (2004)).
(11) Blood Sample Assays
Plasma glucose levels were determined using a glucose B test (Wako Pure Chemical Industries), while plasma insulin levels were determined using insulin immunoassay (Shibayagi).
(12) Hyperinsulinemic Euglycermic Clamp Study
Clamp test was carried out as described in the following documents (J. Biol. Chem. 281, 26602-26614 (2006); J. Biol. Chem. 281, 8748-8755 (2006)), with slight modifications. Two or three days before the test, an infusion catheter was inserted into the right jugular vein under general anesthesia with sodium pentobarbital. The test was performed on mice under conscious and unstressed conditions after 6-hr fasting. A primed continuous infusion of insulin (Humulin R, Lilly) was given (3.0 milliunits/kg/min) and the blood glucose level, monitored every 5 min, was maintained at 120 mg/dl for 120 minutes by administration of glucose (5 g of glucose per 10 ml enriched to 20% with [6,6-2H2] glucose (Sigma)). Blood was sampled via tail tip after 90, 105, and 120 minutes for determination of the rate of glucose disappearance (Rd). The Rd was calculated according to nonsteady-state equations and endogenous glucose production (EGP) was calculated as a difference between Rd and exogenous glucose infusion rate (J. Biol. Chem. 281, 26602-26614 (2006); J. Biol. Chem. 281, 8748-8755 (2006)).
(13) Exercise Therapy
The treadmill exercise test was conducted according to the regimen of 15 m/min for 30 min a day for 2 weeks.
(14) RNA Interference
Two pairs of siRNAs were chemically synthesized, annealed, and transfected into C2C12 myocytes by using Lipofectamine RNAiMAX (Invitrogen) (J. Biol. Chem. 278, 2461-2468 (2003)). The sequences of the sense siRNAs are as follows:
AMPKα1, AMPKα2, CaMKKβ, PGC-1α, SIRT1, and LKB1 siRNA were purchased from Applied Biosystems. Cells transfected with nonfunctional-jumbled siRNA (unrelated) (Applied Biosystems) were used as negative controls. Fourth eight hours after transfection, the cells were lysed.
(15) NAD+/NADH Measurements
NAD+ and NADH nucleotides were directly measured by the method described in the following document (Nature, 458, 1056-1060 (2009)). Whole skeletal muscles or two 10 cm dishes of C2C12 myocytes were homogenized in 200 μl of an acid extraction buffer to measure the NAD+ concentration, or 200 μl of alkali extraction to obtain the NADH concentration. Next, homogenates were neutralized and the concentration of nucleotides was measured fluorimetrically after an enzymatic cycling reaction using 5 μl of a sample. Values for both nucleotides were detected within the linear range. NAD+/NADH ratios were calculated by comparing the ratios obtained from each animal or from parallel cell dishes in each experiment.
(16) Calcium Imaging in C2C12 Myocytes
C212 myocytes cultured on glass base dishes (IWAKI) were treated with 2.5 μM fura-2/AM for 30 min. Fluorescence was measured with an Aquacosmos Calcium Imaging system (Hamamatsu Photonics). Ligands were delivered through the superfusing Ringer's solution (140 mM NaCl, 5.6 mM KCl, 5 mM HEPES, 2.0 mM sodium pyruvate, 1.25 mM KH2PO4, 2.0 mM CaCl2, 2.0 mM MgCl2, 9.4 mM D-glucose (pH 7.4)). For the calcium chelating experiment, EGTA solution (140 mM NaCl, 5.6 mM KCl, 5 mM HEPES, 2.0 mM sodium pyruvate, 1.25 mM KH2PO4, 2.0 mM MgCl2, 5.0 mM EGTA-2Na, 9.4 mM D-glucose (pH 7.4) was used for the bath solution. Fura-2 fluorescence ratios were calibrated to calcium ion signals.
(17) Gene Expression in Xenopus Oocytes and Electrophysiological Recording
Stage V to VII oocytes were treated with 2 mg/ml of collagenase S-1 (Nitta gelatin) in calcium ion-free saline solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) for 1 to 2 hr at room temperature. The resulting oocytes were then microinjected with 50 ng of cRNA for mAdipoR1. The cRNA was synthesized from linearized modified pSPUTK. The injected oocytes were incubated for 3 days at 18° C. in a bath solution (88 mM NaCl, 1 mM KCl, 0.3 mM Ca(NO3)2, 0.4 mM CaCl2, 0.8 mM MgSO4, 2.4 mM NaHCO2, 15 mM HEPES, pH 7.6) supplemented with 10 μg/ml of penicillin and streptomycin.
Whole cell currents were recorded with a two-electrode voltage clamp technique. Intracellular glass electrodes were filled with 3M KCl. Signals were amplified with an OC-725C amplifier (Warner Instruments), low-pass filtered at 50 Hz, and digitized at 1 kHz. A control bath solution contained 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES. It was titrated to pH 7.2 with NaOH. A calcium ion free solution containing 1.8 mM BaCl2 instead of CaCl2 was used. The inward current was monitored at a holding potential of −80 mV. Calcium ion activated Cl− current was monitored by applying 500-mscc depolarizing pulses of +100 mV and +50 mV from the holding potential of −80 mV. Adiponectin was supplied to the superfusing bath solution via a silicon tube connected to computer-driven solenoid valves. Data acquisition and analysis were carried out using Digidata 1322A and pCLAMP software.
(18) Calcium Imaging in Skeletal Muscle
From 8-10 weeks-old mice, soleus muscles were dissected. Preparations were treated with 5 μM fura-2/AM in the presence of 0.1% Chremophore EL (Sigma) for 15 min. Fluorescence images were acquired using an inverted fluorescence microscopy (IX71, Olympus) equipped with a cooled charge-coupled device (CCD) camera (iXon, Andor) and a dry objective lens (10×, NA=0.3, Olympus) through a filter set consisting of a 327-353 nm and 369-391 nm excitation filter, a 409 nm dichroic mirror, and a 515-560 nm fluorescent filter. Ligands were supplied using the superfusing Ringer's solution (150 mM NaCl, 4 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 30 mM D-glucose, 5 mM HEPES (pH 7.4)).
(19) Insulin Resistance Index
Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were conducted by the method described in the following documents (Nature Med. 8, 1288-1295 (2002); Nature Med. 7, 941-946 (2001)), with some modifications. The areas of the glucose and insulin curves were calculated, respectively, by multiplying the cumulative mean height of the glucose values (1 mg/ml=1 cm) and insulin values (1 ng/ml=1 cm) by time (60 min=1 cm) (Nature Med. 13, 332-339 (2007); Nature Med. 7, 941-946 (2001)). The insulin resistance index was calculated from the product of the areas of glucose and insulin×10−2 in the glucose tolerance test. The results were expressed as the percentage of the value of control littermates.
(20) Citrate Synthase Activity
Citrate synthase activity was determined from the homogeneates by using a spectrophotometer according to the method described in the following documents (Cell Metab. 4, 75-87 (2006); Eur. J. Biochem. 10, 198-206 (1969); Obes Res 13, 574-581 (2005)). The citrate synthase activity was measured at 37° C. in 0.1M Tris-HCl (pH 8.3) assay buffer containing 0.12 mM 5,5′-dithio-bis(2-nitrobenzoic acid) and 0.6 mM oxaloacetate. After recording of absorbance at 412 nm in the first two minutes, the reaction was started with the addition of 3 mM acetyl-CoA and a change in absorbance was measured every 10 sec for 7 min.
(21) Oxygen Consumption
Oxygen consumption of animals under the fasting condition was measured using an O2/CO2 metabolism measurement system every 3 min for 24 hr (Model MK-5000; Muromachi Kikai, Tokyo, Japan). Each mouse was placed in a hermetically sealed chamber (560 ml volume) with an air flow of 0.5 l/min. The oxygen consumed was converted to milliliters/minute by multiplying it by the flow rate.
(22) Increased Calcium Influx Experiment
Hydrogels were prepared by crosslinking aqueous gelatin solutions with glutaraldehyde according to the method described in the following document (J. Control Release 64, 133-142 (2000)). For incorporating Bay-K 8644 into the resulting gelatin hydrogels, 20 μl of Bay-K 8644 (0.25 μM) was added dropwise onto 20 mg of freeze-dried hydrogel and left it overnight at 4° C. Under anesthesia, the gelatin hydrogel having Bay-K 8644 incorporated therein was implanted under the skin. Then, the skin was carefully sutured with No. 4-0 nylon monofilament.
(23) Patch Clamp Test
Whole-cell currents were amplified with a patch-clamp amplifier (CEZ-2400; Nihon Kohden) and were digitized with a PowerLab digitizer (AD Instruments). Ringer's solution was used for the extracellular solution. The electrode solution contained 140 mM KCl, 10 mM HEPES, 5 mM EGTA-2K, and 10 mM D-glucose (pH 7.4). The data were low-pass filtered at 2 kHz and were sampled at 20 kHz. Ligands dissolved in Ringer's solution were applied at least twice with a self-made pressure-ejection system and via a two-barreled glass capillary.
(1) Decreased PGC-1α and Mitochondria in Muscle-R1KO Mice
Muscle-R1KO mice showed decreased phosphorylation of AMPK (
In the muscle-R1KO mice, a decrease in the molecules involved in mitochondrial biogenesis such as estrogen-related receptor α (Esrra) (Proc. Natl. Acad. Sci. USA 101, 6570-6575 (2004)), the molecules involved in transcription such as nuclear respiratory factor 1 (Nrf1), and the molecules involved in mitochondrial DNA replication/translation such as mitochondrial transcription factor A (Tfam) was observed (
The mitochondrial function was assessed by measuring the enzymatic activities of Cox (
(2) Decreased Type I Muscle Fibers and Exercise Capacity in Muscle-R1KO
In muscle-R1KO mice, the molecules involved in type I muscle fiber (Physiol. Rev. 80, 1215-1265 (2000)) differentiation myocyte enhancer factors 2C (Mef2c) (EMBO J. 19, 1963-1973 (2000)) (
To study the impact of AdipoR1 ablation on skeletal muscle function in intact animals, muscle endurance test by tread mill running for assessing the involuntary physical exercise was conducted in control and muscle-R1KO mice. The muscle endurance of muscle-R1KO mice was significantly lower than that of the control mice (
In muscle-R1KO mice, the expression levels of both mitochondrial and cytoplasmic oxidative stress-detoxifying genes such as manganese superoxide dismutase (Sod2) (
(3) Insulin Resistance in Muscle-R1KO Mice
The glucose levels and plasma insulin levels after administration of glucose were significantly higher in muscle-R1KO mice than in control mice (
In agreement with the data obtained from the hyperinsulinemic euglycermic clamps, decreased tyrosine phosphorylation of IRS-1 (
(4) CaMKKβ is Required for Adiponectin/AdipoR1-Induced PGC-1α
Next, the molecular mechanisms by which muscle-R1KO mice showed the above-mentioned phenotypes were studied. Incubation of C2C12 myocytes added with 30 μg/ml adiponectin increased a mitochondrial DNA content (
Suppression of the expression of AdipoR1 or CaMKKβ by respective specific siRNAs (
It has been reported that PGC-1α is activated after phosphorylation by AMPK (Proc. Natl. Acad. Sci. USA 104, 12017-12022 (2007)) or activated by deacetylation through SIRT1 activation (Nature 434, 113-118 (2005)). Therefore, the possibility of AMPK, which had been activated by adiponectin and AdipoR1, regulating PGC-1α activities was studied. Treatment of C2C12 myocytes with adiponectin decreased PGC-1α acetylation after treatment for 2 hr (
In C2C12 myocytes expressing PGC-1α-R13 in which 13 of the potential acetylation sites of lysine was mutated into arginine (Nature 434, 113-118 (2005)), adiponectin failed to further induce mitochondrial biogenesis (
SIRT1 deacetylase activity has been reported to depend on the NAD+ levels (Nature 434, 113-118 (2005); Nature 444, 868-874 (2006)). Adiponectin increased a NAD+/NADH ratio in C2C12 myocytes (
(5) Adiponectin Induces Calcium Ion Influx Via AdipoR1.
Interestingly, treatment of C2C12 myocytes with adiponectin increased an intracellular calcium ion concentration as measured by fura-2-based fluorescent imaging (
In order to study further the importance of AdipoR1 in adiponectin-induced calcium ion responses in the gain of function study system, AdipoR1 was expressed in Xenopus oocytes (
(6) Adiponectin-Induced Calcium Ion Influx is Important for Action Expression.
Incubation of C2C12 myocytes after addition of adiponectin thereto increased the phosphorylation of the AMPK α-subunits at Thr 172 and EGTA partially suppressed an increase in AMPK phosphorylation induced by adiponectin (
Suppression of CaMKKβ or LKB1 expression by respective siRNAs specific to them (
Whether adiponectin-induced calcium ion influx into skeletal muscle decreased in muscle-R1KO mice or not was studied by in vivo imaging (Am. J. Physiol. Heart Circ. Physiol. 275, H1652-H1662 (1998); J. Biol. Chem. 281, 1547-1554 (2006)). Treatment of soleus muscle with adiponectin increased the intracellular calcium ion concentration in control mice as measured by fura-2-base fluorescent imaging. On the other hand, calcium responses of soleus muscle to adiponectin almost completely disappeared in muscle-R1KO mice (
Whether or not simultaneous activation of calcium ion signaling pathway and AMPK/SIRT1 pathway by exercise could rescue phenotype in muscle-R1KO mice independent of AdipoR1 was studied. Two weeks' exercise significantly ameliorated insulin resistance and increased mitochondrial content and function such as citrate synthase activities in the muscle of muscle-R1KO mice (
The results will next be described in detail.
(7) Muscle-R1KO Deteriorates Adiponectin Action in Skeletal Muscle.
Muscle-R1KO exhibited decreased phosphorylation of AMPK (
(8) Decreased Type I Muscle Fibers in Muscle-R1KO
Muscle-R1KO exhibited a decrease in molecules involved in type I muscle fiber differentiation myocyte enhancer factor 2C (Mef2c) (EMBO J. 19, 1963-1973 (2000)) (
(9) Protein Expression Levels of MEF2, MCAD, SOD2, Catalase, and Glut4 in the Muscle of Muscle-R1KO Mice
The protein expression levels of MEF2, MCAD, SOD2, catalase, and Glut4 in the muscle of muscle-R1KO mice were significantly decreased compared with those seen in control mice (
(10) Increased Amount of Mitochondria in C2C12 Myocytes Treated with Adiponectin
Incubation of C2C12 myocytes with 30 μg/ml adiponectin increased a mitochondrial DNA content and amount of mitochondria as assessed by using MitoTracker green (
(11) NAD+ and NADH Contents in C2C12 Myocytes and Skeletal Muscle
Adiponectin increased the NAD+/NADH ratio and NAD+ and NADH contents in C2C12 myocytes (
(12) PPARα Levels and Activity in Skeletal Muscle Of Muscle-R1KO Mice
Muscle-specific disruption of AdipoR1 had no significant effect on PPARα (Ppara) levels (
(13) Oxygen (O2) Consumption and Respiratory Quotient (RQ) of Muscle-R1KO Mice
Muscle-specific disruption of AdipoR1 significantly decreased O2 consumption (
(14) The mRNA Levels of Glut 4 in Gastrocnemius and Soleus Muscles
The expression levels of Glut4 (Slc2a4) were significantly decreased in muscles rich in type I muscle fibers such as soleus muscle and also muscles rich in type II fibers such as gastrocnemius of muscle-R1KO as compared with those in control mice (
(15) The Effect of Increased Calcium Influx in Muscle of Muscle-R1KO Mice
An increase in CaMKKβ activity caused by the addition of 0.25 μM Bay-K 8644, a calcium ion channel opener (Nature 303, 535-537 (1983); Am. J. Physiol. 258, R462-468 (1990)), (
(16) The Effect of Exercise, AICAR, Resveratrol, and Antioxidant MnTBAP on Muscle-R1KO Mice
Administration of AICAR (Am. J. Physiol. 273, E1107-1112 (1997)) significantly increased the phosphorylation of AMPK in the muscle of muscle-R1KO mice (
Although MnTBAP (Nature 440, 944-948 (2006)) indeed significantly decreased ROS such as H2O2 (
AICAR significantly but partially rescued the AdipoR1 loss in muscle (
(17) Mitochondrial Capacity and Insulin Signaling
Suppression of Adipor1 by specific siRNA (
(18) FOXO1 Phosphorylation in Skeletal Muscle of Muscle-R1KO Mice
Insulin-stimulated phosphorylation of Thr 24 and Ser 253 in Foxo (EMBO J. 19, 989-996 (2000)) was markedly decreased in the skeletal muscle of muscle-R1KO mice as compared with that in control mice (
(19) The Amounts of PGC-1α Acetylation in Adiponectin-Stimulated C2C12 Myocytes Treated with Deacetylase SIRT1 Knockdown
The amounts of PGC-1α acetylation in adiponectin-stimulated C2C12 myocytes treated with deacetylase SIRT1 knockdown were almost the same as those in C2C12 myocytes without adiponectin stimulation. These results show the possibility that under the conditions of the present inventors such as 6-hr fasting, deacetylase SIRT1 may not be sufficiently activated in C2C12 myocytes without adiponectin stimulation (
(20) Inward Current Responses to Adiponectin
Inward current responses to adiponectin were observed by whole-cell patch-clamp recordings of C2C12 myocytes at a holding potential of −60 mV (
Stimulation of oocytes expressing AdipoR1 with adiponectin induced inward current responses (
(21) Phosphorylation of AMPK Stimulated with Adiponectin in the Presence of BAPTA-AM or U73122
Bis-(o-aminophenoxy)ethane-N,N,N9,N9-tetra-acetic acid, tetraacetoxymethyl ester (BAPTA-AM) (intracellular calcium chelator) partially suppressed adiponectin-induced increased AMPK phosphorylation to almost the same extent as EGTA (
(22) Sequential Phosphorylation of CaMKI, AMPK, and HDAC5 and their Extracellular Calcium Ion Influx Dependency
Treatment of C2C12 myocytes with adiponectin caused a rapid, robust, and transient increase in CaMKI phosphorylation (J. Biol. Chem. 273, 31880-31889 (1998); Trends Biochem. Sci. 24, 232-236 (1999)) (
Next, whether adiponectin stimulates phosphorylation of histone deacetylase (HDAC) 5 subsequent to the activation of CaMKI (Nature 408, 106-111 (2000)) and AMPK (Diabetes 57, 860-867 (2008)) was examined. CaMK and/or AMPK signaling prevent formation of MEF2-HDAC complexes and induce nuclear export of HDAC5 by phosphorylation of this transcriptional repressor (Nature 408, 106-111 (2000); Diabetes 57, 860-867 (2008)). These signaling pathways may be mechanisms by which CaMK and/or AMPK increases PGC-1α expression via MEF2 in myocytes. The treatment of C2C12 myocytes with adiponectin increased HDAC5 phosphorylation in a sequential and partial manner depending on extracellular calcium ion influx (
(23) Comparison Between Insect Olfactory Receptors and AdipoR1
It has been reported that insect olfactory receptors lack homology to GPCR, possess an opposite seven-transmembrane topology with the amino terminus located intracellularly and induce extracellular calcium ion influx (Nature 452, 1002-1006 (2008)). There is therefore the possibility that signaling by AdipoR1 is similar to signaling of insect olfactory receptors. The above-mentioned results suggest the development of new anti-diabetic and anti-atherogenic drugs with AdipoR1 as a molecular target.
(24) Differences Between PGC-1α and Adiponectin/AdipoR1 in the Regulation of Insulin Sensitivity
PGC-1α is a key regulator of oxidative metabolism in skeletal muscle. Reduced PGC-1α and OXPHOS levels have been associated with insulin resistance and type 2 diabetes in humans (Nature Genet. 34, 267-273 (2003)) and mice (
(25) Conclusion
As described above, muscle-R1KO mice exhibited a decreased mitochondrial content and enzyme. PGC-1α is a key regulator of a mitochondrial content and function. It has been reported that PGC-1α expression is modulated in several physiological conditions. For example, in skeletal muscle, it is modulated by increased calcium ion signaling via molecules such as CaMK and CREB in partial response to exercise (Nature 454, 463-469 (2008)). It has also been reported that PGC-1α activities are modulated by several kinds of PGC-1α modifications, such as phosphorylation via AMPK (Proc. Natl. Acad. Sci. USA 104, 12017-12022 (2007)) and deacetylation via AMPK and SIRT1 (Nature 434, 113-118 (2005)). AMPK and SIRT1 could also be activated by exercise. It has been proved that muscle-R1KO mice exhibited decreased PGC-1α expression as well as decreased PGC-1α acetylation. Consistent with it, it has also been proved that adiponectin induces calcium ion influx via AdipoR1 and thereby activates CaMKKβ and causes increased PGC-1α expression and at the same time, adiponectin/AdipoR1 activates AMPK and SIRT1, which induces deacetylation of PGC-1α. These data suggest that adiponectin and AdipoR1 stimulate increases in both the expression and activation of PGC-1α in a similar manner to exercise (
Although the decreasing degree of PGC-1α expression was approximately 40% in muscle-R1KO mice, the decreasing degrees of mitochondrial biogenesis and exercise endurance were comparable to those observed in muscle-specific PGC-1α-knockout mice (J. Biol. Chem. 282, 30014-30021 (2007)), which may be explained by the finding that adiponectin and AdipoR1 increase not only PGC-1α expression but also PGC-1α activities (
Decreases in the expression of AdipoR1 and PGC-1α and mitochondrial DNA content were also observed in type 2 diabetic patients (Nature Genet. 34, 267-273 (2003)) and individuals at increased risk of developing diabetes due to their family history (Proc. Natl. Acad. Sci. USA 100, 8466-8471 (2003)) as well as in obese diabetic db/db mice (
In skeletal muscle, AdipoR1 is involved in regulation of insulin sensitivity via multiple mechanisms (
It has been reported that exercise has beneficial effects on longevity and life-style related diseases, and at the same time on activation of calcium ions, AMPK, SIRT1, and PGC-1α pathways (Nature 454, 463-469 (2008)). It has been demonstrated that adiponectin and AdipoR1 regulate PGC-1α and mitochondria via calcium ions and AMPK/SIRT1. Therefore, the agonism of AdipoR1 as well as strategies of increasing AdipoR1 in muscle could be pseudo-exercise effect.
In conclusion, AdipoR1 plays a critical role in the physiological and pathophysiological significance of adiponectin in muscle and are involved in the regulation of calcium ion signaling, PGC-1α expression and activation, mitochondrial function and oxidative stress, glucose and lipid metabolism, and exercise endurance. The above-mentioned results suggest that the agonism of AdipoR1 as well as strategies of increasing AdipoR1 in muscle may provide a new treatment method for mitochondrial dysfunction, insulin resistance, and type 2 diabetes linked to obesity.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/056043 | 3/15/2011 | WO | 00 | 5/15/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61314280 | Mar 2010 | US |
